Rabbit anti atf4

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Rabbit anti-ATF4 is a primary antibody that specifically recognizes the activating transcription factor 4 (ATF4) protein. ATF4 is a transcription factor that plays a role in the cellular stress response and various cellular processes.

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Anti-ATF4 Rabbit anti-TM

5 protocols using rabbit anti atf4

Retinal Cell Protein Expression Analysis

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Retinas and cells were lysed in radioimmune-precipitation-assay buffer with a protease-inhibitor mixture, phenylmethylsulfonyl fluoride and sodium orthovanadate (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein mixtures (25 μg per well) were resolved by SDS-PAGE and transferred to membranes, which were probed with mouse anti-TNF-α, anti-VEGF, anti-phospho-JNK (phosphorylated c-Jun N-terminal kinase; p-JNK) and anti-HIF1-α (hypoxia-inducible transcription factor 1α) antibodies, rabbit anti-ATF4 (activating transcription factor 4; these antibodies were obtained from Santa Cruz Biotechnology and used at 1:200 dilution), rabbit anti-GRP78 (78 kDa glucose-regulated protein; Abcam, Cambridge, MA, USA; 1:2000), anti-phospho-eIF2α (phosphorylated eukaryotic translation initiation factor 2α; p-eIF2α; Cell Signaling, Technology, Danvers, MA, USA; 1:1000) and anti-ATF6 (Abcam; 1:1000). Each membrane was stripped and reblotted with a mouse anti-β-actin antibody (Abcam; 1:10,000), to demonstrate similar levels of the loading control β-actin in each lane.

Yang J., Chen C., McLaughlin T., Wang Y., Le Y.Z., Wang J.J, & Zhang S.X. (2019). Loss of X-box binding protein 1 in Müller cells augments retinal inflammation in a mouse model of diabetes. Diabetologia, 62(3), 531-543.

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Chromatin Immunoprecipitation in Pancreatic Cells

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For immunoprecipitation, Min6 cells or isolated mouse islets were washed 3 times with cold 1x PBS, then nuclear extract was collected as previously described [20] except buffer C did not contain glycerol. Nuclear lysate was incubated overnight with antibodies for denoted proteins or control IgG and then incubated for 2 h with G Protein Dynabeads (ThermoFisher). For ChIP, Min6 cells (∼1 × 10⁷ cells) or 200 isolated mouse islets were cross-linked with 1% formaldehyde in 1xPBS for 10 min at room temperature and quenched with glycine to a final concentration of 0.125 M. Chromatin was sonicated in a Bioruptor (Diagenode, Denville, NJ) and precleared with normal mouse IgG (Santa Cruz) overnight at 4 °C. After removal of an aliquot for input, precleared chromatin was divided equally for IP with either rabbit anti-ATF4 (Santa Cruz), anti-HA conjugated agarose (Sigma), or normal IgG (Santa Cruz) for 3 h at 4 °C. Use and dilution information for anti-sera is found in Table 1.

Juliana C.A., Yang J., Cannon C.E., Good A.L., Haemmerle M.W, & Stoffers D.A. (2018). A PDX1-ATF transcriptional complex governs β cell survival during stress. Molecular Metabolism, 17, 39-48.

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Protein expression analysis by WB and FC

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The western blot antibodies include rabbit anti-ATF4 (Santa Cruz), mouse anti-TATA-box binding protein (Thermo Pierce), mTOR substrates antibody sampler kit (9862; Cell Signaling Technology). For flow cytometric analysis of pS6, anti-pS6 (Alexa Fluor 488; Cell Signaling Technology) was used.

Yang X., Xia R., Yue C., Zhai W., Du W., Yang Q., Cao H., Chen X., Obando D., Zhu Y., Chen X., Chen J.J., Piganelli J., Wipf P., Jiang Y., Xiao G., Wu C., Jiang J, & Lu B. (2018). ATF4 Regulates CD4+ T Cell Immune Responses through Metabolic Reprogramming. Cell reports, 23(6), 1754-1766.

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Protein Lysate Preparation and Western Blot

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Protein lysates were prepared using NP-40 lysis buffer (0.4% NP-40, 150 mM NaCl, 50 mM Tris base, pH 7.6). Protein concentration was determined using BCA (ThermoFisher, Saint Louis, Missouri). Proteins were separated by SDS-PAGE and immunoblotted with the following primary antisera: rabbit anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA), mouse anti-tubulin (Sigma Aldrich), rabbit anti-Atf4 (Santa Cruz), guinea pig anti-Pdx1 (Chris Wright), and anti-HA-HRP conjugated (Roche, San Francisco, CA, USA). HRP conjugated antibodies were incubated for 2 h at room temperature. Luminate Crescendo HRP substrate (Millipore) was used to visualize blots with film or a Chemi-doc Touch Imaging system (Bio-Rad, Hercules, California, USA). Use and dilution information for anti-sera is found in Table 1.

Juliana C.A., Yang J., Cannon C.E., Good A.L., Haemmerle M.W, & Stoffers D.A. (2018). A PDX1-ATF transcriptional complex governs β cell survival during stress. Molecular Metabolism, 17, 39-48.

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Protein expression analysis by WB and FC

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